1. Technical Field
This invention relates to a method and apparatus for the analysis of a sample, and particularly though not exclusively to fluorescence analysis.
2. Related Art
Fluorescence analysis is an important experimental tool for both chemists and biologists, and is of particular interest to pharmaceutical researchers.
A known method of analysing fluorescence comprises exciting a sample of many hundreds of fluorophores with an intense pulse of light from a laser. The intensity of fluorescence emitted by the sample at a given delay following excitation is detected. The sample is excited again, and the intensity of fluorescence emitted by the sample at a different delay following excitation is detected. A series of measurements at various delays are made, and the fluorescence intensity is plotted as a function of the delay, to provide a distribution of fluorescence intensity over time. An upper state lifetime characteristic of the fluorophores comprising the sample may be derived from the gradient of the intensity distribution (for example, by fitting an exponential decay curve to the distribution).
Various other methods of measuring characteristic fluorophore lifetimes are known, and in each case the phase shift or demodulation of a detected signal compared to an excitation signal must be measured (see for example J R Lacowitz, Principles of Fluorescence Spectroscopy, Plenum Press, New York & London). However, present methods require comparatively lengthy averaging processes, which are unsuited to modern high speed processing of many measurements (for example in high throughput screening, or imaging).
A further limitation of the above method is that it requires a sample of many hundreds of fluorophores and high intensity illumination. A sample of the order of 100 fluorophores will not provide sufficient signal to noise to allow the measurement of fluorescence. The method cannot therefore be used to measure effects caused by a single fluorophore or a sample containing a small number of fluorophores or a sample that optically quenches rapidly. Furthermore, the method requires the use of pulsed laser sources and gated detectors to measure time domain fluorescence parameters such as decay rate.
A further known technique, known as number fluctuation spectroscopy may be used to determine the diffusion coefficients of particles in a fluid. Number fluctuation spectroscopy comprises illuminating a specified volume of fluid which particles will diffuse into and out of, and detecting light scattered by the particles when they are within the specified volume. An auto-correlation of the detected scattered light with itself will yield a characteristic frequency which is a function of the diffusion rate of the particles within the fluid. Number fluctuation spectroscopy may be used to determine a particle mean diffusion coefficient and thus may be used to determine a particle size.
WO96/27798 describes a technique known as fluorescence correlation spectroscopy, which may be considered to be a variant of number fluctuation spectroscopy. The technique is used when there are other scattering particles in the fluid which are not required to contribute to the detected signal. The particles of interest are made to fluoresce, and the fluorescent light is spectrally separated from the scattered light so that only the diffusion of the particles of interest is measured. Fluorescence correlation spectroscopy allows the determination of a diffusion coefficient, but does not provide the measurement of fluorescent lifetimes.